Abstract The assembly of the vitamin K-dependent complexes of blood coagulation on cell surfaces expressing phosophatidylserine is responsible for explosive thrombin formation following vascular damage. Long-standing dogma backs a preeminent role of the activated platelet in this process coupled with contributions from tissue factor-bearing cells. In contrast, the vascular endothelium is considered to play an inhibitory role by the facilitation of the anticoagulant reactions to limit thrombus growth. This clear-cut demarcation in the opposing roles played by activated platelets and endothelium has increasingly been called into question by recent findings in vivo. The goal of Project 1 is to investigate the contributions of platelets and the vascular endothelium in supporting coagulation enzyme assembly and function. We will exploit our expertise in developing novel site-specific fluorescent derivatives of the clotting proteins for biophysical studies to now permit quantitative imaging of bound coagulation proteins in vitro and in vivo. Under Aim 1, we will employ flow chamber studies using site-specific fluorescent derivatives of Xa, Va and prothrombin for quantitative imaging of the interaction of the constituents of prothrombinase with activated endothelial cells and adherent platelets. We also propose fluorescence lifetime imaging studies on these cells with appropriate fluorescence donor- acceptor pairs designed to assess the distribution of productively assembled enzyme. Aim 2 will extend these approaches to studies in vivo using the mouse cremaster laser injury model. High speed confocal intravital fluorescence imaging will be employed for quantitative analysis of bound prothrombinase constituents following infusion of site-specific fluorescent derivatives of recombinant mouse proteins. We also propose pharmacologic and genetic approaches to further dissect the relative contributions of adherent platelets versus damaged endothelium in supporting prothrombinase assembly and function. In aim 3, we bring new advances in the understanding of the enzymology of meizothrombin and prothrombin activation to bear on a previously unappreciated but important regulatory reaction in coagulation. In this aim, we propose to address how meizothrombin, with a selectively anticoagulant spectrum of activities of thrombin may regulate the hemostatic response by virtue of its production as an intermediate of prothrombin activation on the activated endothelium but not on platelets. We bring powerful approaches to bear on major but unanswered questions in blood coagulation biology to derive new insights into the mechanisms by which blood coagulation may be spatially regulated in vivo.